Automatic isothermal titration microcalorimeter apparatus and method of use

ABSTRACT

Automated isothermal titration micro calorimetry (ITC) system comprising a micro calorimeter with a sample cell and a reference cell, the sample cell is accessible via a sample cell stem and the reference cell is accessible via a reference cell stem. The system further comprises an automatic pipette assembly comprising a syringe with a titration needle arranged to be inserted into the sample cell for supplying titrant, the pipette assembly comprises an activator for driving a plunger in the syringe, a pipette translation unit supporting the pipette assembly and being arranged to place pipette in position for titration, washing and filling operations, a wash station for the titrant needle, and a cell preparation unit arranged to perform operations for replacing the sample liquid in the sample cell when the pipette is placed in another position than the position for titration.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/326,300, filed on Dec. 2, 2008, the contents of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to microcalorimeters and morespecifically to features that improve the performance ofmicrocalorimeters, especially an automated isothermal titration microcalorimetry system (ITC system).

Microcalorimeters are broadly utilized in fields of biochemistry,pharmacology, cell biology, and others. Calorimetry provides a directmethod for measuring changes in thermodynamic properties of biologicalmacromolecules. Microcalorimeters are typically two cell instruments inwhich properties of a dilute solution of test substance in an aqueousbuffer in a sample cell are continuously compared to an equal quantityof aqueous buffer in a reference cell. Measured differences between theproperties of the two cells, such as temperature or heat flow, areattributed to the presence of the test substance in the sample cell.

One type of microcalorimetcr is an isothermal titration calorimeter. Theisothermal titration calorimeter (ITC) is a differential device, butoperates at a fixed temperature and pressure while the liquid in thesample cell is continuously stirred. The most popular application fortitration calorimetry is in the characterization of the thermodynamicsof molecular interactions. In this application, a dilute solution of atest substance (e.g., a protein) is placed in the sample cell and, atvarious times, small volumes of a second dilute solution containing aligand, which binds to the test substance, are injected into the samplecell. The instrument measures the heat, which is evolved or absorbed asa result of the binding of the newly introduced ligand to the testsubstance. From results of multiple-injection experiments, properties,such as, the Gibbs energy, the association constant, the enthalpy andentropy changes, and the stoichiometry of binding, may be determined fora particular pairing between the test substance and the ligand.

While currently utilized ITCs provide reliable binding data results,their widespread utilization in the early stages of drug developmenthave been limited by several factors: the relatively high amounts ofprotein required to perform a binding determination (e.g., about 0.1milligram (mg) to about 1.0 mg of a protein), the limited throughput dueto the time required to perform the measurement and the complexity ofusing conventional ITCs.

Today, gathering binding data utilizing prior art ITCs require extensivepreparation and skill by the practitioner. For example, using prior artITCs, the reference and sample cells are first filled respectively withthe reference substance and sample substance via a corresponding cellstem. Next, a titration pipette of the ITC is filled with a titrant,which is a delicate operation as it is very important that the syringein the pipette is accurately filled and that there is no air trappedtherein. Then a needle of the titration pipette is manually placed inthe sample cell via the cell stem, and the ITC experiments can beinitiated. The ITC measurement procedure is controlled by a control unitin the form of a computer or the like running a program for performingthe experiments. Consistent with the program used for the experiment, astirring motor rotates the syringe, needle, and paddle at an assignedspeed enabling proper mixing of the reagents. Consistent with theprogram used for the experiment (e.g., when a certain temperature and/orequilibrium are reached), a plunger in the syringe is activated toinject the titrant into the sample solution. The injection can be donediscretely (step-by-step) or continuously, depending on the programsettings. The calorimeter continuously measures and records the heatrelease/absorption versus time associated with the interaction ofreagents. The analysis of the results is done according to theestablished algorithm.

As would be appreciated by a reading of the above-described prior artprocedure, utilizing prior art ITCs, the quality of binding measurementsperformed with these prior art ITCs depends heavily of the operator'sskills and experience, and involves a considerable amount of preparationtime.

For some time there has been at least one automated ITC system on themarket, MicroCal AutoITC, which is based on a commercially availablemicro calorimeter and a linear robot system and a fluidics systemarranged to perform automatic sample handling.

SUMMARY OF THE INVENTION

The object of the invention is to provide a new automatic isothermaltitration micro calorimetry system (ITC system), which ITC systemovercomes one or more drawbacks of the prior art. This is achieved bythe ITC system as defined in the independent claims.

One advantage with the present ITC system is that each titrationexperiment requires less time compared to the prior art. This is e.g.due to the reduced cell volume and that washing and refilling of thepipette assembly and the sample cell is performed essentially inparallel. Hence the system throughput is considerably higher compared tothe prior art systems, making it possible to evaluate large number ofsamples to make screening type experiments.

Another advantage is that the ITC system may be arranged to perform alarge number of unattended titration experiments.

Embodiments of the invention are defined in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic example of a prior art manual ITC system incross-section, the ITC system comprising an automatic pipette assembly.

FIG. 2 shows a schematic view of one embodiment of an automated ITCsystem.

FIGS. 3 a and 3 b show schematic views of other embodiments of theautomated ITC system.

FIGS. 4 a and 4 b show schematic views of two embodiments of a syringefluidics system for an automated ITC system.

FIGS. 5 a to 5 c show schematic views of the function of a syringe fillport connector unit according to an embodiment.

FIG. 6 shows a schematic view of a cell preparation fluidics systemaccording to one embodiment.

FIG. 7 schematically shows different states of operations for the ITCsystem of FIG. 2

FIGS. 8 a to 8 d shows different states of operations for the ITC systemof FIG. 2 more in detail.

DETAILED DESCRIPTION OF THE INVENTION

In the PCT application PCT/US2008/081961, which is incorporated byreference, a manual ITC system 10 of the type disclosed in FIG. 1 ispresented. According to one embodiment, the manual ITC system 10 isutilized as micro-calorimeter in the present automatic ITC system, butin other embodiments, the micro-calorimeter is of other types, as willbe discussed in greater detail below. In the disclosed manual ITC system10, the cell compartment volume is reduced by about a factor of seven ascompared to prior art ITCs, without a reduction in sensitivity, and witha significantly faster response time. Such an ITC system permits theperformance of experiments with about 10 times less protein sample, andwith only a total of about 2 to about 4 titrations per hour. In additionto reducing the costs associated with running the ITC experiment, asmaller cell volume also extends the number of ITC applications. Forexample, the range of binding affinities that can be measured by ITC isdictated by a parameter called “c value,” which is equal to the productof the binding affinity (K_(a)) and the total concentration (M_(total))of macromolecule (c=[M_(total)]F_(d)). For accurate affinitydetermination, the c value must be between 1 and 1,000. A decrease inthe cell volume by a factor of ten results in a similar increase in cvalue if the same amount of protein is used, and, consequently, theability to measure weak binders. This ability is especially important inthe early stages of drug discovery, in which binding affinities areweak, especially in conjunction with a fully automated instrument.

FIG. 1 schematically shows one embodiment of the manual ITC system 10that may be automated in accordance with the present invention. The ITCsystem 10 comprises a micro calorimeter 20 and an automatic pipetteassembly 30. The micro calorimeter 20 comprises a reference cell 40 anda sample cell 50 which are designed to be essentially identical in heatcapacity and volume. The cells 40 and 50 are comprised of a suitablechemically inert and heat conductive material, such as gold, Platinum,tantalum, hastelloy or the like. The cells 40 and 50 may be ofessentially any suitable shape, but it is desirable that they are of thesame shape, that they are possible to arrange in a fully symmetricarrangement, and that efficient mixing of the titrant with the samplemay be achieved. In the disclosed embodiment, the cross-section of thecells 40 and 50 is rectangular, and the cross-section in the transversehorizontal direction may be circular, resulting in coin shaped cellswith circular facing surfaces.

In order to reduce any external thermal influences to a minimum, the,reference cell 40 and the sample cell 50 are both enclosed by a firstthermal shield 60 which in turn is enclosed by a second thermal shield70. The thermal shields 60, 70 may be comprised of any suitablethermally conductive material such as silver, aluminum, supper or thelike. The shields 60, 70 may further be comprised of one or morethermally interconnected sub shields (not shown, to provide even furtherstable temperature conditions for the calorimetric cells 40, 50.

In order to control the temperature of the shields 60, 70, thermalcontrol means may be arranged to control the temperature thereof. In anITC system said thermal control means are mainly used to set the“isothermal” temperature of the calorimeter, ie of the thermal shields60, 70, before the titration experiments are initiated. But as will bedisclosed in greater detail below, said thermal control means may alsobe used to improve the adiabatic behavior of the calorimeter. Accordingto one embodiment, the thermal control means are comprised of one ormore heat pump units, such as a thermoelectric heat pump device based onthe peltier effect or the like. Other types of thermal control meansinclude thermostatically controlled liquid baths, mechanical heat pumps,chemical heating or cooling systems or the like.

In the disclosed embodiment a first heat pump unit 80 is arranged totransfer heat energy between the first 60 and second thermal shields 70,a second heat pump unit 90 is arranged to transfer heat energy betweenthe second thermal shield 70 and a heat sink 100 in thermal contact withthe ambient temperature. A temperature controller 110 is arranged tocontrol the first and second heat pump units 80, 90 so that the desiredtemperature conditions are achieved. The temperature controller 110monitors the temperatures of the first 60 and second thermal shield byassociated temperature sensors 120 and 130 respectively. Furthermore,the thermal controller 110 is arranged to control the cell temperatureby a cell heating arrangement 145. The thermal controller 110 iscontrolled via a calorimeter user interface run on a computer 150 or thelike. Calorimetric sensors 140 for sensing the temperature differencebetween the sample cell 50 and reference cell 40 during the ITCexperiments may be connected to the computer 150, e.g. via apreamplifier 160.

A reference cell stem 170 and a sample cell stem 180 provides access tothe reference cell 40 and sample cell 50, respectively, for supplyingreference and sample fluids, titration fluid, washing of the cells etc.In the disclosed embodiment, the cell stems 170 and 180 both extendsessentially vertically through both thermal shields and the heat sink toprovide direct communication with cells 40 and 50 and the cell stems 170and 180 each support their respective cell 40 and 50 in the cavity ofthe first thermal shield 60.

The automatic pipette assembly 30 comprises a pipette housing 190, asyringe 200 with a titration needle 210 arranged to be inserted into thesample cell 50 for supplying titrant, and a linear activator 220 fordriving a plunger 230 in the syringe 200. The titration needle 210 isrotatable with respect to the housing 190 and is provided with astirring paddle 240 arranged to stir sample fluid in the sample cell 50in order to achieve efficient mixing of titrant and sample fluid. Theautomatic pipette assembly 30 further comprises a stirring motor 250 fordriving the rotation of the titration needle 210.

In the embodiment disclosed in FIG. 1 the stirring motor 250 is a directdrive motor with a hollow rotor arranged concentric with the syringe 200and the titration needle 210. The syringe 200 is at its upper endsupported for rotation by the stirring motor 400 and at the lower end bya bearing 260.

In an alternative embodiment, not shown in the figures, the stirringmotor 250 drives the titration needle for rotation by a rotationtransmission arrangement, such as a drive belt arrangement, a drivewheel arrangement or the like. Moreover, the stirring motor may bearranged separated from the pipette assembly 30 and be arranged to drivethe titration needle for rotation by a suitable transmission arrangementsuch as a magnetic coupling or the like.

The automatic pipette assembly 30 is controlled by a controller of theITC system, e.g. stirring of the sample and the titration.

In the disclosed embodiment, the linear activator 220 comprises astepper motor 270 arranged to drive the threaded plunger 230 thatextends coaxially through the hole of a hollow rotor and into thesyringe 200 wherein it is rotatably attached to a pipette tip 280 thatseals against the inner wall of the syringe 200 to allow displacing aprecise volume of titration liquid from syringe 200. The linearactivator 220 may be of any other type capable of perform controlledlinear motion with sufficient precision. This design allows syringe tobe rotated independently of the main body 190 of the pipette assembly30; at the same time, the linear activator 220 can drive the threadedplunger 230.

In accordance with one embodiment, schematically disclosed in FIGS. 2 to8 d, there is provided an automated isothermal titration microcalorimetry (ITC) system 300 comprising:

-   -   a micro calorimeter 20 with a sample cell 50 and a reference        cell 40, the sample cell 50 is accessible via a sample cell stem        180 and the reference cell 40 is accessible via a reference cell        stem 170,    -   an automatic pipette assembly 30 comprising a syringe 200 with a        titration needle 210 arranged to be inserted into the sample        cell 50 for supplying titrant, the pipette assembly 30 comprises        a linear activator 220 for driving a plunger 230 in the syringe        200,    -   a pipette translation unit 310 supporting the pipette assembly        30 and being arranged to place pipette in position for        titration, washing and filling operations,    -   a wash station 320 for the titrant needle 210, and    -   a cell preparation unit 330 arranged to perform operations for        replacing the sample liquid in the sample cell 50 when the        pipette 30 is placed in another position than the position for        titration.

The micro calorimeter 20 may be of any type capable of performing ITCcalorimetric measurements using sufficiently small volumes of samplesuch as the micro calorimeter 20 schematically shown in FIG. 1. Asdisclosed above (FIG. 1), but not specifically shown in FIGS. 2 to 8 d,a micro calorimeter generally comprises a sample cell 50 and a referencecell 40, wherein the sample cell 50 is accessible via a sample cell stem180 and the reference cell 40 is accessible via a reference cell stem170 (shown as circular openings in FIG. 2). The automatic pipetteassembly 30 may be of the type disclosed above, but it may be of anysuitable design comprising a syringe 200 with a titration needle 210arranged to be inserted into the sample cell 50 for supplying titrant.Like above, the pipette assembly 30 may further comprise a linearactivator 220 for driving a plunger 230 in the syringe 200. However, thesyringe 200 may be of essentially any type, capable of providingwell-defined volumes of titrant. The titration needle 210 may berotatable and may be provided with a paddle 240 for stirring of theliquid in the sample cell 50 during the titration. The stirring may beaccomplished as is discussed above or in any other suitable way.

The pipette translation unit 310 may be of any type capable of placingthe pipette in the appropriate positions for titration, washing andfilling. FIGS. 2 and 3 schematically show two different types oftranslation units, wherein FIG. 2 shows a rotation translation unit 310and FIG. 3 shows a linear translation unit 310 b. In order to place(insert) the titration needle 210 in position in the sample cell 50,and/or in other positions, the pipette translation unit 310 is capableof moving the pipette 30 in the vertical direction with respect to themicro calorimeter 20. The pipette translation unit 310 may bemechanically restricted with respect to its freedom of movement so thatit only may move between mechanically predetermined positions, or it maybe a general translation unit of robot type that is restricted tomovement between said predetermined positions by means of softwareparameters, or a combination thereof. For clarity reasons no such meansfor vertical movements have been included in the FIGS. 2 to 8 d.

The wash station 320 is arranged at a suitable position wherein thetitration needle 210 of the pipette assembly 30 can be placed inposition for washing. The wash station 320 may be of any suitable typecapable of washing at least the section of the titration needle 210 thatis immersed in the sample during titration when the pipette assembly 30is placed in position for washing. According to one embodiment, the washstation 320 comprises a wash cavity 340 arranged to receive thetitration needle. The wash station 320 is made of any suitable materialthat is inert with respect to the reagents used in the ITC experimentsand the wash cycles. According to one embodiment, the wash station 320comprises a waste outlet port 350 at the bottom end of the wash cavityconnected to a waste removal unit 360. The waste outlet port 350 is usedto remove waste liquids as well as wash liquids during the pipettewashing cycle, as will be disclosed more in detail below, and it ispreferably arranged at the bottom end of the wash cavity 340 in order toenable complete drainage of the wash cavity. In one embodiment notdisclosed in the figures, the pipette translation unit 310 is limited tomovement in the vertical direction, and the wash station 320 instead isarranged to be moved to a position in alignment with the needle 240 forcleaning of the same.

In FIGS. 2 to 8 d the cell preparation unit 330 is shown as atranslation unit of the same type as the pipette translation unit 310,but arranged to be positioned in at least two positions related towashing and replacing sample liquid in the sample cell. By the provisionof a cell preparation unit 330 for replacing the sample liquid in thesample cell 50 the total cycle time is reduced and thus the throughputof the ITC system 300 is increased, as the sample cell 50 may be washedand filled with new sample liquid at the same time as the pipette 30 iswashed and filled with new titrant.

FIG. 2 schematically discloses an automated isothermal titration microcalorimetry (ITC) system 300 according to one embodiment of the presentinvention. As mentioned above, the translation units 310, 330, 370 inthis embodiment are all of rotary type, and all positions of operationare arranged along circular paths of the rotary translation units. Inanother embodiment, not shown, one or more of the rotary translationunits are provided with additional linear translation means to extendwork area and to increase the flexibility.

In FIG. 2 the pipette translation unit 310 comprises a pipette arm 380that is rotatably supported for rotation about an axis A, and supportingthe pipette assembly 30 at the other end thereof. The pipette arm 380 isfurther arranged to move the pipette vertically, either in that the arm380 can be moved vertically along the axis A, or in that the arm 380 islimited for rotation in one plane and the pipette 30 is verticallymoveable with respect to the arm 380. The pipette arm 380 is arranged toplace pipette 30 in position for:

-   -   titration with the titration needle inserted into the sample        cell 50,    -   washing and filling with the titration needle inserted in a        combined wash/fill station 320.

The combined wash/fill station 320 may be a wash station of the typediscussed above with an outlet port 350 at the bottom end of the washcavity 340. The outlet port 350 is connected to a waste fluidics system360 that will be discussed in more detail below.

The cell preparation unit 330 is in turn comprised of a correspondingcell arm 390 that is rotatably supported for rotation about an axis B,supporting a cell cannula 400 connected to a cell fluidics system 410for dispensing and withdrawing liquid in the sample cell 50 andpotentially also in the reference cell 40. The cell fluidics system 410will be disclosed in more detail below. The cell arm 390 is arranged tomove the cell cannula 400 to a plurality of positions such as the cells,40, 50 of the micro calorimeter, one or more sample sources, and asample preparation station 420, or the like. In the disclosedembodiment, four different sample source positions are included of whichthree positions represent large volume sample reservoirs of vial type430 a-430 c, e.g. for standard sample liquids, and the fourth positionan autosampler position 440, e.g. for specific or sensitive sampleliquids, wherein the cell cannula 400 is arranged to draw the sampleliquid from a specific well in a sample tray 450 (e.g. micro plate orthe like). In the disclosed embodiment, the autosampler position 440 isa static position to which the cell cannula 400 can be moved by the cellarm 390 and be lowered into a specific well of a sample tray 450 thatcan be moved to position a selected well at the autosampler position 440by a tray actuator (not shown). The tray actuator may be of any suitabletype capable of selectively position a specific sample well of a sampletray 450 at a desired position, such as a linear X-Y actuator or arotary actuator with a carousel tray. The sample preparation station 430may be used to prepare the sample before it is transferred into the cell50 or 40, e.g. by bringing the sample to a temperature close to theexperimental temperature, or by degassing through mixing.

The ITC system 300 disclosed in FIG. 2 further comprises a titranttransfer unit 370 arranged to transfer titrant from a primary titrantsource, e.g. a sample tray 450, to the wash/fill station 320. In thedisclosed embodiment, the transfer unit 370 comprises a titrant transferarm 460, e.g. corresponding to the cell transfer arm 380, that isrotatably supported for rotation about an axis C, supporting a transfercannula 470 connected to a syringe fluidics system 480. The titranttransfer arm 460 is arranged to position the titrant cannula 470 in anautosampler position 440 for drawing a titrant sample from a titrantwell in a sample tray 450, and in position to dispense said titrantsample in the wash/fill station 320. The autosampler position 440 andthe sample tray 450 may be a separate position and tray with respect tothe cell cannula autosampler position 440 discussed above, but as isdisclosed in FIG. 2, the cell cannula 470 and the titrant cannula 400may be positioned at the same autosampler position 440 (not at the sametime) and the tray actuator may be controlled to position appropriatewells for the respective cannula at the autosampler position 440. Thesyringe fluidics system 480 is further connected to a fill portconnection unit 490 being arranged to selectively connect to a fill port500 at an upper section of the syringe 200 in the pipette assembly. Whenconnected to the fill port 500, the fill port connection unit 490provides fluidic contact between the syringe cavity and the syringefluidics system 480 to selectively pull or push liquid or gas throughthe syringe 200.

As previously mentioned, FIG. 3 a shows an ITC system corresponding tothe system of FIG. 2, but wherein the translation units 310 b, 330 b,370 b for the pipette 30, cell cannula 400 and transfer cannula 470 areof linear type, and the associated positions of operations are arrangedaccordingly. Moreover, the cell arm 390 b and the transfer arm 460 b aremoveable in two dimensions (disregarding the vertical direction asmentioned above) whereby one or both may be controlled to position theassociated cannula in a selected well in a static sample tray 450. FIG.3 b shows an embodiment of a linear ITC-system similar to FIG. 3 a,wherein the titrant transfer unit is omitted and the pipette translationunit 310 c is arranged to place the pipette 30 in position for fillingdirectly from a selected well in the sample tray 450. Moreover, the fillport connection unit 490 is arranged by the pipette 30 on the pipettearm 380 c in order to connect to the fill port 500 both when the pipette30 is placed in the wash station 320 and in a fill position in a well ofthe sample tray 450.

As mentioned above, the waste fluidics system 360 is connected to theoutlet port 350 of the wash station 320 for withdrawing fluid from thewash station. According to one embodiment, the waste fluidics system 360comprises a waste pump 510 for selective withdrawal of fluid from thewash station 320, optionally in combination with one or morecontrollable valves 520 to direct the flow of waste fluids. In otherembodiments, the waste pump may be a common pump for one or morefluidics systems in the ITC system 300, and one or more valves maycontrol the flow in the systems, respectively. The waste pump 510 may beany suitable pump capable of removing the fluids in the wash station,such as a peristaltic pump, a syringe pump or the like. FIG. 4 a shows aschematic view of an embodiment of a waste fluidics system 360comprising a waste pump 510 of reservoir type, such as a syringe pump,and a waste control valve 520 for selective connection disconnection ofthe waste pump to the outlet port 350, a waste outlet 530 and a ventport 540.

As mentioned above, and shown more in detail in FIGS. 4 a to 5 c, thesyringe 200 of the pipette may comprise a fill port 500 at an uppersection thereof, providing fluidic contact with the syringe cavity whenthe plunger 230 is positioned above said fill port 500. Further the ITCsystem 300 may comprise a mating fill port connection unit 490 beingarranged to selectively connect to the fill port 500, thereby providingfluidic contact between the syringe cavity and a syringe fluidics system480 arranged to selectively pull or push liquid or gas through thesyringe as part washing and filling operations, which will be disclosedmore in detail below. As is schematically disclosed, the fill port 500may be a bore through a wall of the syringe 200, and the bore may be ofany suitable shape such as straight or conical. The connection unit 490comprises a connection member 550 mating shape and/or of resilientmaterial to achieve a reliable and fluid tight connection. The syringefluidics system 480 may further be connected to the transfer cannula470, and arranged to control aspiration and dispensing of fluids duringtitrant transfer operations, as well as washing operations of thetransfer cannula 470.

According to one embodiment, the syringe fluidics system 480 comprises afill pump 560 to selectively pull or push liquid in the fluidics system,optionally in combination with one or more controllable valves 570, 580,590 to direct the flow of fluids and a purge gas source 600. In otherembodiments, the fill pump may be a common pump for one or more fluidicssystems in the ITC system 300, and one or more valves may control theflow in the systems, respectively. The waste pump 560 may be anysuitable pump capable of push or pull the liquids in the syringe fluidicsystem with relatively high accuracy, such as a peristaltic pump, asyringe pump or the like. FIG. 4 a shows a schematic view of anembodiment of a syringe fluidics system comprising a fill pump 560 ofreservoir type, such as a syringe pump, a syringe control valve 570,syringe purge valve 580 and a transfer cannula purge valve 590. Thesyringe control valve 570 provides selective connection disconnection ofthe fill pump 560 to the fill port 500 of the syringe 200, to thetransfer cannula 470, to a plurality of reagent reservoirs 610 a-d, to awaste outlet 620 and to a vent port 630. The reagent reservoirs 610 a-dmay comprise wash liquids for washing the syringe 210 and/or thetransfer cannula 470 or the like. FIG. 4 b shows a schematic view ofanother embodiment of the syringe fluidics system 480 and the wastefluidics system 360, wherein the transfer cannula 470 is not connectedto the syringe fluidics system 480, but instead connected to the wastefluidics system 360, whereby the titrant sample is transferred from thetransfer cannula 470 to the wash station 320 via the waste fluidicssystem and the outlet port 350 of the wash station. Further, FIGS. 4 aand 4 b schematically show the pipette assembly 30 in position fortitration, with the titration needle 210 inserted into the sample cell50.

In some embodiments, as is previously discussed, the syringe 200 isrotatable with respect to the automatic pipette 30 and is driven forrotation by a stirring motor 250. Then, in order to locate the positionof the fill port 500, the fill port connection unit 490 may comprise aport alignment mechanism 640 arranged to prevent rotation of the syringeat a predetermined angular position when a connection member 550 of theconnection unit is aligned with the fill port 500. FIGS. 5 a to 5 cschematically shows an example of an alignment mechanism 640, whereinthe syringe 200, or any other part that is arranged to rotate with thesyringe 200, is provided with an alignment member 650, and the fill portconnection unit 490 is provided with a rotation stop unit 660 that maybe actuated to interfere with the rotation path of the alignment member650, and when the alignment member 650 abuts the rotation stop unit 660,then the connection member 550 is aligned with the fill port 500. Thealignment procedure comprises the steps of:

-   -   actuating the stop unit 660 (FIG. 5 a),    -   rotating the syringe slowly in a predetermined direction until        further rotation is prevented by the stop unit 660 abutting the        alignment member 650 (FIG. 5 b), and    -   actuating the connection member 550 to connect to the fill port        500 (FIG. 5 c).

The stop unit 660 and the connection member 550 may be actuated byelectromagnetic drive actuators in the form of an electric motorarrangement, a solenoid or the like, or they may be actuated by ahydraulic or pneumatic actuator or the like capable of moving the stopunit 660 and the connection member 550. In order to achieve a fluidtight connection between the syringe fill port 500 and the syringefluidics system 480, the connection member 550 is pressed against thefill port 500 with a predetermined force. In one embodiment (not shown)the connection member 550 is actuated by an electromagnetic driveactuator, to move the connection member 550 into contact with the fillport and the connection member 550 is spring loaded with respect to theactuator, whereby the scaling force is determined by the spring constantand the compression of said spring.

As is e.g. disclosed in FIG. 2, the fill port connection unit 490 may bearranged at the wash station 320 to enable connection between thesyringe cavity and the syringe fluidics system 480 when the pipetteassembly 30 is arranged at the wash station 320. But, as is disclosed inFIG. 3 b, the fill port connection unit 490, may be arranged togetherwith the pipette assembly 30 supported by the pipette translation unit310, whereby fluidic connection between the syringe fill port 500 andthe syringe fluidics system 480 may be established in any position ofoperation.

According to one embodiment, the ITC system 300 is arranged to utilizethe syringe fill port 500 to wash the syringe 200 and the titrationneedle 210 by pushing one or more wash liquids through the syringe 200and the titration needle 210 via the syringe fill port 500 when thetitration needle 210 of the pipette 30 is arranged in the wash cavity340 of the wash station 320. Thereafter the system may dry the syringe200, needle 210 and the wash station 320 by purging gas through thesyringe 200 and the titration needle 210 via the syringe fill port 500after washing the same.

In many situations it is important to fill the syringe 200 of thepipette assembly 30 with titrant without having any trapped air in thesyringe 200. In one embodiment, this is achieved by pulling apredetermined volume of titrant into the syringe from a titrant source,e.g. the wash station 320, in which the titration needle 210 isinserted, wherein the predetermined volume is selected to be larger thanthe syringe volume, whereby the syringe is overfilled and the titrantstart to exit the syringe through the fill port 500. Then the linearactivator 220 is activated to close the fill port 500 by moving theplunger 230 below to the fill port 500.

FIG. 6 schematically shows an example of a cell fluidics system 410connected to the cell cannula 400 for dispensing and withdrawing liquidin the sample cell 50 and potentially also in the reference cell 40. Asis previously discussed, the cell cannula 400 may be arranged to bepositioned in the sample cell 50, in a well of a sample tray 450, in oneor more large volume sample reservoirs 430 a-c and a sample preparationstation 420. According to one embodiment, the cell fluidics system 410comprises a cell pump 670 for selective dispensing and withdrawing offluid through the cell cannula 400, optionally in combination with oneor more controllable valves 680, 690 to direct the flow of cell washfluids or the like. In other embodiments, the cell pump 670 may be acommon pump for one or more fluidics systems 360, 380, 410 in the ITCsystem 300, and one or more valves may control the flow in the systems,respectively. The cell pump 670 may be any suitable pump capable ofdispensing and withdrawing the fluids in e.g. the sample cell 50, suchas a peristaltic pump, a syringe pump or the like. FIG. 6 shows aschematic view of an embodiment of a cell fluidics system 410 comprisinga cell pump 670 of reservoir type, such as a syringe pump, a cellpreparation control valve 680 for selective connection disconnection ofthe waste pump to the cannula 400, four cell wash liquid reservoirs 700a-c, a waste outlet 710 and a vent port 720, and a purge select valve690 for connection of the cannula 400 to the cell preparation valve 680or a source of purge gas 600 for drying the cannula 400.

FIG. 7 schematically shows the ITC system of FIG. 2 wherein positions ofoperation for each translation arm is shown by broken lines.

FIGS. 8 a to 8D schematically show examples of states wherein operationsfor preparation of the pipette and the sample cell may be performed inparallel in the ITC system of FIG. 2.

In FIG. 8 a the pipette assembly 30 is placed at the wash position withthe titration needle 210 in the wash station 320 for a syringe washcycle. During a wash cycle, the connection member 550 of the fill portconnection unit 490 is connected to the fill port 500 of the syringe200, and the syringe fluidics system 480 is arranged to push and pullone or more washing liquids through the syringe 200 optionally followedby purging a gas, e.g. nitrogen, through the syringe to dry the syringe200. To push a wash liquid through the syringe, firstly the syringecontrol valve 570 is arranged in position A to connect the fill pump tothe appropriate reagent reservoir 610 a-d and the fill pump 560 isactuated to draw wash liquid into its pump reservoir, secondly thesyringe control valve 570 is arranged in position B to connect the fillpump 560 to the fill port 500 of the syringe 200 and the fill pump 560is actuated to push wash liquid through the syringe 200, whereby thewash liquid is dispensed from the titration needle 210 into the washstation 320. A full syringe cleaning cycle might comprise pushing thesame or a different wash liquid (syringe valve positions C-E) two ormore times through the syringe 200, and it may further involve pullingliquid from the wash station 320 through the syringe 200 and into thepump reservoir whereby it can be pushed through the syringe 200 one moretime, or be discarded through the waste outlet 620 of the syringefluidics system 480. When the fill pump 570 is arranged to consecutivelypush two or more different wash liquids through the syringe 200, thefill pump 570 may be rinsed to avoid contamination between washingliquids, by filling the pump reservoir with a rinse liquid, e.g. water.Liquid that is dispensed into the wash station 320 may be selectivelywithdrawn through the outlet port 350 by the waste pump 510 into thepump reservoir by setting the waste valve 520 in position A, and maythereafter be discarded through the waste outlet 530 by setting thewaste valve 520 in position B.

In FIG. 8 a, while the syringe wash cycle is performed at the washstation 320, the titrant transfer unit 370 is arranged to draw titrantsample from a well in the sample tray 450 by setting the syringe valve570 in position F and pulling titrant from the well using the fill pump560. The operation of drawing titrant from the well may e.g. beperformed when the syringe 200 is purged with dry gas, whereby the fillpump 560 and the syringe valve 570 are not involved in the wash cycle,but after the fill pump 560 and the syringe valve 570 has beenthoroughly rinsed and washed to avoid contamination.

In FIG. 8 a, also while the syringe wash cycle is performed at the washstation 320, the cell preparation unit 330 is arranged to remove theprevious sample from the sample cell 50 and to wash the sample cell 50.As the pipette assembly 30 is removed from the sample cell 50, the cellcannula 400 may be inserted into the sample cell 50, and by arrangingthe cell preparation control valve 680 in position A the cell pump 670may be activated to withdraw the previous sample into the pumpreservoir, and the previous sample may thereafter be discarded throughthe waste outlet 710 by setting the cell preparation control valve 680valve in position B. Cleaning of the cell is thereafter performed bydispensing and withdrawing one or more cell wash liquids in the samplecell 50 optionally followed by purging a gas, e.g. nitrogen, through thecell cannula to dry the sample cell 50. To dispense a wash liquid in thesample cell, firstly the cell preparation control valve 680 is arrangedin position C, D, E or F to connect the cell pump 670 to the appropriatewash liquid reservoir 700 a-d and the cell pump 670 is actuated to drawwash liquid into its pump reservoir, secondly the cell preparationcontrol valve 680 is arranged in position A to connect the cell pump 670to the cannula 400 and the cell pump 670 is actuated to dispense washliquid in the sample cell 50 through the cell cannula 200. The cleaningliquid(s) are thereafter withdrawn and discarded through the waste port710 of the cell preparation control valve 680. According to thedisclosed embodiment, the cell cannula 400 stays in the sample cellduring the cell wash procedure, thereby the cell cannula 400 is washedat the same time as the cell 50 and is ready for transfer of freshsample liquid to the sample cell 50.

In FIG. 8 b the pipette assembly 30 is placed at an intermediateposition between the sample cell 50 and the wash station 320, in orderto grant the titrant transfer cannula 470 of the titrant transfer unit370 access to the wash station 320 to dispense a new titrant sampletherein, and to grant the cell cannula 400 cell preparation unit 300access to the sample cell 50 to fill the sample cell with fresh samplein the next step. The titrant transfer unit 370 is arranged to dispensethe titrant sample from the cannula 470 into the wash station by settingthe syringe valve 570 in position F and dispensing titrant using thefill pump 560.

In FIG. 8 b the cell preparation unit is arranged to draw fresh samplefrom a well in the sample tray 450 by inserting the cell cannula 400 ina selected well containing the desired fresh sample, setting the cellpreparation control valve 680 in position A and pulling fresh samplefrom the well into the pump reservoir of the cell pump 670.Alternatively, the cell cannula 400 may be inserted into one of thesample reservoirs 430 a-c.

In FIG. 8 c the pipette assembly 30 again is placed at the wash positionwith the titration needle 210 in the wash station 320 to fill thesyringe 200 with titrant. As is discussed in detail with reference toFIGS. 5 a-c, during filling of the syringe 200, the connection member550 of the fill port connection unit 490 is connected to the fill port500 of the syringe 200, and the syringe fluidics system 480 is arrangedto pull the titrant through titrant needle 210 into the syringe 200until a small volume has passed the fill port 500, whereby the plunger230 is lowered to close the fill port 500. By drawing the titrant intothe syringe in this way, trapped air in the titrant is effectivelyavoided. During the state disclosed in FIG. 8 b, the titrant transferunit 370 is essentially inactive, but the cell preparation unit 330 ispositioned with the cell cannula 400 in the sample cell 50 to fill thelater with an exact amount of fresh sample by arranging the cellpreparation control valve 680 in position A and activating the cell pump670 to dispense the fresh sample contained in the pump reservoir intothe sample cell 50.

In FIG. 8 a the pipette assembly 30 is placed at the titration positionwith the titration needle 210 in the sample cell 50 to perform an ITCexperiment. The Titration transfer unit 370 is now positioned with thetitrant cannula in the wash station 320 to wash it before the nexttitrant transfer operation. The washing cycle may be essentially thesame as for the syringe 200. During the state disclosed in FIG. 8 b, thecell preparation unit 330 is essentially inactive, and shown with thecell cannula 400 in the sample preparation station 420.

Examples of Liquid Handling Sequences Includes:

Cell Wash:

-   -   a. The cell cannula 400 is inserted into the cell 40, 50,        resting on the bottom.    -   b. The cell content is drawn through the cell cannula 400, into        the cell pump 680, and dispensed out to the waste port 710.    -   c. Water is drawn from one of the wash liquid reservoirs 700 a-d        into the pump reservoir of the cell pump 680 and dispensed out        to the waste port 710 to rinse the syringe.    -   d. A wash liquid is drawn into the pump reservoir of the cell        pump 680 from one of the wash liquid reservoirs 700 a-d and        dispensed through the cell cannula 400 into the cell 40, 50 in        the exact amount needed to fill the cell,    -   e. The wash liquid is cycled back and forth from the pump        reservoir of the cell pump 680 to the cell 40, 50 to wash the        later.    -   f. The steps beginning at step b are repeated a predetermined        number of times until the cell 40, 50.    -   g. The cell is emptied to waste as in step b.    -   h. The cell cannula 400 is moved to the sample preparation        station 420 and dried with by purging gas, e.g. Nitrogen. If        degassing is included in the cell load procedure, the sample        preparation 420 station is cleaned before drying.

Pipette Wash:

-   -   a. The pipette 30 is placed in the wash/fill station 320 and the        fill port 500 is connected.    -   b. The pipette plunger 230 is raised above the fill port 500        allowing liquid to flow through the syringe 200 of the pipette        30.    -   c. First water then air is dispensed from the syringe pump 560        into the fill port 500, through the syringe 200 and titration        needle 210 into the wash/fill station 320. Simultaneously, this        water is drawn in great excess from the bottom of the wash/fill        station 320 through the waste outlet 350 into the waste pump        510.    -   d. The waste pump 510 is stopped and a precise amount of water        is dispensed through the pipette 30 to fill the wash/fill        station 320 to the top of the outside of the titration needle        210. The water is cycled back and forth to wash the entire        syringe 200 and the titration, inside and outside.    -   e. Step c is repeated.    -   f. Step d is repeated with methanol.    -   g. Step c is repeated.    -   h. Nitrogen is purged through the fill port 500 to dry the        system.    -   i. The pipette 30 is removed from the wash/fill station 320 to        allow the titrant transfer unit 370 to load the station 320 with        titrant sample.    -   j. The syringe pump 560 is rinsed with water to clear any        methanol from the system.

Cell Load:

-   -   a. Titrant sample is drawn into the cell cannula 400 from a        sample tray 450 or a sample reservoir 430 a-c. It is then slowly        dispensed into the cell to prevent air bubbles. Optionally, the        sample is dispensed into the sample preparation station 420 to        be warmed and mixed (degassed) before being transferred into the        cell 50.

Pipette Load:

-   -   a. Titrant is drawn into the titrant transfer cannula 470 from a        sample tray 450 and dispensed into the wash/fill station 320.    -   b. The pipette 30 is placed in the wash/fill station 320 and the        fill port 500 is connected.    -   c. The plunger 230 is raised above the fill port 500 allowing        liquid to flow through the pipette.    -   d. A precise volume of titrant is drawn up through the titration        needle 210 by the syringe pump 560, over-filling the syringe 200        such that a small amount of titrant exits the fill port 500.    -   e. The plunger 230 is lowered below the fill port 500 leaving        the titrant needle 210 and pipette syringe 200 completely        filled.

Titrant Transfer Clean:

-   -   a. The titrant transfer cannula 470 is placed in the wash/fill        station 320 and is rinsed with water, then rinsed with methanol        and dried in much the same way that the syringe 200 of the        pipette is washed and dried.

It is to be understood that any feature described in relation to any oneembodiment may be used alone, or in combination with other featuresdescribed, and may also be used in combination with one or more featuresof any other of the embodiments, or any combination of any other of theembodiments. Furthermore, equivalents and modifications not describedabove may also be employed without departing from the scope of theinvention, which is defined in the accompanying claims.

1-17. (canceled)
 18. An automated isothermal titration micro calorimetry(ITC) system arranged to perform a plurality of unattended titrationexperiments in series, the system comprising: a system controller forcontrolling the automated operation of the ITC system; a microcalorimeter comprising a sample cell and a reference cell, the samplecell being accessible via a sample cell stem and the reference cellbeing accessible via a reference cell stem; an automatic pipetteassembly comprising a syringe with a titration needle arranged to beinserted into the sample cell for supplying titrant, the pipetteassembly comprising an activator for driving a plunger in the syringecavity; a wash station; a sample fill station; a pipette translationunit supporting the pipette assembly that places a pipette in positionfor titration with the titration needle in the sample cell and inposition for washing operations with the titration needle in the washstation; a cell preparation unit that performs operations for replacingthe sample liquid in the sample cell when the pipette is placed inanother position than the position for titration.